Diagnostic compositions for aminoarylsulphonamides



Patented May 25, 1943 UNlTED STATES PATENT OFFICE DIAGNOSTICCOMPOSITIONS FOR AMINO- ARYLSULPHONAMIDES Jonas Kamlet, Brooklyn, N. Y.,assignor to Miles Laboratories, Inc., Elkhart, Ind., a corporation ofIndiana No Drawing. Application October 20, 1941,

Serial No. 415,773

14 Claims.

convenient method by means of which the average physician or nurse mayperform these tests with a. high degree of accuracy and specificitywithout the use of complicated laboratory equipment or specializedanalytical training.

Another object of this invention is to provide a single simple test bymeans of which all of these sulpha" compounds can be detected andestimated with a good degree of accuracy.

Since the discovery that sulfanilamide is effective in the treatment ofbaterial infections due to the hemolytic streptococcus, gonococcus,meningococcus, pneumococcus'and related organisms, this compound, aswell as numerous derivatives thereof, has become one of themcstimportant chemotherapeutic agents in medical practice. Foremostamong the suipha drugs most widely employed at present aresulphanilamide, sulphanilylamidopyridlne (sulphapyridine),sulphanilylamidothiazole (sulphathiazole), sulphanilylamidopyrimidine(sulphadiazine), sulphanilylguanidine, sulphanilylsulphanilamlde(disulon) and 4-(4-amino-benzenesulphon)amidobenzenesulphondimethylamide (Uliron). New derivatives ofsulphanilamide, each adapted to a specific chemotherapeutic function,are being .introduced into the medical armamentarium' with considerablefrequency.

The group of compounds referred to as aminoarylsulphonamides in thisspecification has the general formula.

NHr-O-SOaNlELR centration in the blood stream of the patient. Because ofindividual variations in the rates of absorption and excretion, noempirical rule for dosage is absolutely reliable. If an adequatebacteriostatic concentration of the drug is not obtained, it cannotfulfill its therapeutic function. If the physician is unaware of thisfact, he may not administer the greater dose that is required and thusfurther imperil his patient.

Inadequate doses of an aminoarylsulphonamide may also serve to build upa drug-resistant strain of the pathogenic microorganisms in the body ofthe patient which even adequate doses of the drug subsequentlyadministered may fail to control. (Schmidt, Claugus and Starks,Proceedings of the Society for Experimental Biology and Medicine, 45,256, October 1940.) On the other hand, excessively high concentrationsof the drug in the blood stream are distinctly dangerous. The literaturereports numerous-undesirable sequelae, such as acidotic cyanoses,methemeglobinemias and blood dyscrasias such as agranulocytoses, oftenterminating fatally after excessive doses of sulpha drugs. There is alsoa greater tendency for the slightly soluble conjugated (i. e.,acetylated) aminoarylsulphonamides to precipitate in the urinary tractwhen large doses of the drug are given over a period of time. Theseprecipitated particles serve as the nuclei of urinary concretions andcause bloody and painful urination long after the administration of thesulpha" drug has'been discontinued.

It is generally believed now that the optimum bacteriostaticconcentration in the blood is between 7.0 and 10.0 mgm. per cent forsulphanilamide, and between 4.0 and 7.0 mgm. per cent forsulphapyridine. When the concentration of either of these drugs exceeds15.0 m'gm. per cent, their further administration should bediscontinued. As a rule, it is usually sufiicient to attain the desiredblood concentration of the aminoarylsulphonamide and then to continuethe same dosage until the patient has shown the desired response. In themeantime, the excretion of the drug in the urine can be followed byfrequent determinations.

If the rate at which the drug is being excreted in the urine shouldsuddenly diminish or increase, another blood determination is indicated.

Thus, with occasional blood determinations and more frequent urineanalyses, the aminoarylsulphonamide concentration in the blood can bekept within the optimum range throughout the whole course of treatment.

The methods heretofore employed for the detection and estimation ofaininoarylsulphonamides in body fluids require not only some technicalability and experience, but also make use of equipment and chemicalswhich are not always available to the average medical practitioner. Mostof these methods are based on the diazotization of theaminoarylsuphonamide and the coupling of the resultant diazoniumcompound with a.,i:'hromophoric component to produce a highly coloredazo dye. Fuller (Lancet, 1, 194, 1937) coupled diazotized sulphanilamidewith thymol or B-naphthol in alkaline solution, but the resultant colorwas unsuitable for accurate colorimetric comparison. Marshall, Emersonand Cutting (Journal of the American Medical Association, 108, 953,1937) and Kamlet (Journal of Laboratory and Clinical Medicine, 23, 1101,1938) described the use or dimethyl-alphanaphthylamine as a couplingcomponent in acid solution. Scudi (Journal of Biological Chemistry, 122,539, 1937) advised the use of 4.5-dihydroxy, 2.7-naphthalene disulphonicacid (chromotropic acid) as a chromophore to be coupled with diazotizedsulphanilamide. A recent method by Marshall and Cutting (Bulletin of theJohns Hopkins Hospital; 63, 328, 1938) depends on the diazotization ofthe aminoarylsulphonamide and coupling of the resultant diazoniumcompound, with N-(l-naphthyl) ethylenediamine to form a red azo dye thatcan be estimated colorimetrically. I

All of these methods, however, are entirely unsuitable for use by theaverage -physician as a rapid and simple test that may be performed in arelatively short time in his ofiice, or at the patients bedside. Most ofthem require reagents and apparatus rarely available outside of awellequipped clinical laboratory.

Fortune (U. S. Patent 2,208,096) has described a test foraminoarylsulphonamide compounds in body fluids which is a modificationof' the basic diazotization procedures. Four reagents are used and whichwill react with diluted urine or deproteinized blood filtrate to yielda. color which will be strictly proportional in intensity to theoriginal concentration of aminoarylsulphonamide v therein, and which canbe compared with the color panels of a standard color chart graduated interms of milligrams of aminoarylsulphonamide per 100 cc. of whole bloodor urine.

Werner (Lancet 1939, I, 18) has described a method for the detection andestimation of sulphanilamide in biological fluids requiring a singlereagent only-an aqueous sulphuric acid solution ofp-dimethylaminobenzaldehyde. When a few drops of this reagent are addedto an aqueous fluid (e. g., diluted urine or deproteinized bloodfiltrate) containing sulphanilamide, a yellow color rapidly developsthat is strictly proportional in intensity to the concentration ofsulphanilamide. This color is very stable and suitable for colorimetricanalysis. Werner obtains a sensitivity of 1:500,000 with this method,while Hartman (Journal of Laboratory and Clinical Medicine, 26,

. 401, 1940) who describes a similar method using an aqueoushydrochloric acid solution of p-dimethylaminobenzal dehyde, obtainsequally satisfactory results.

There are two disadvantages to these methods: (a) The necessity of usingstrong mineral acids to keep the p-dimethylaminobenzaldehyde in for thetest. The first reagent effects the deproteinization of the blood sampleand the diazotization of the aminoarylsulphonamide contained therein.The second reagent is added to the deproteinized blood filtrate todestroy the excess of nitrous acid remaining behind after thediazotization. The third reagent contains the chromophore compound,l-amino, 8-naphthol,-3.6- disulphonic acid (H acid) which interacts withthe diazotized aminoarylsulphonamide after the fourth reagent,(containing an alkaline-reacting substance) is added to adjust the pH tothe desired point. After these reagents have been added in the properorder, the color produced in the filtrate is compared with the colorpanels on a standard color chart which is graduated in terms ofmilligrams of the aminoarylsulphonamide per 100 cc. of blood.

Because of the multiplicity of steps involved, the errors introduced bythis method may be of considerable magnitude. The first step (i. e.,protein precipitation with simultaneous diazotization) very often yieldsturbid or colored filtratesamFurthermore, the fiocs of proteinprecipitateithus. produced often tend to enclose mechanically :unreactedportions of the blood. It the protein precipitation is efiectedseparately fromthe'diazotization, the method is further complicatedbyanother step.

It 'is,'therefore, the further purpose of this invention to provide asingle, unitary reagent which can be dispensed in tablet form .or incapsules,

solution. It is impractical to incorporate these mineral acids intosolid compositions, (e. g., tablets or capsules) while solid organicacids (such as oxalic, citric, tartaric, etc.) are unsuitable for thepurpose when used with the ,free base. As a rule, these organiccompounds are not sufilciently acid to keep p-dimethylaminobenzaldehydein solution. (b) The difliculty of making a satisfactory colorcomparison. It is a well known fact among those skilled in the art ofcolorimetry that yellow is the most difiicult color to match forintensity. Even with skilled tech nicians, errors as high as 20% induplicate intensity comparisons of yellow solutions are not unusual. Inpractice, it is almost impossible to obtain satisfactory color panels ofdifferent intensities of yellow.

The basis of the present invention is the finding that compositions ofhigh sensitivity and good specificity for aminoarylsulphonamides can beobtained by mixing.

(a) A salt of a p-di-alkyl-aminobenzaldehyde with a non-oxidizinginorganic acid,

(b) A member of the group consisting of citric acid and tartaric acid,and

(c) A water-soluble blue dyestufi.

Salts of the p-di-alkylaminobenzaldehydes with the non-oxidizinginorganic acids (e. g.,

sulphuric, hydrochloric, phosphoric, etc.) may be prepared by mixing thebase with an aqueous solution of slightly more than an equimolar amountof the acid, and evaporating to dryness. Sulphamic acid cannot be usedas a solvent for the p-di-alkylaminobenzaldehyde since it forms a rdeeply yellow colored water-soluble complex with the base. Oxidizinginorganic acids (such as nitric) cannot be used as a solvent for thep-dialkylaminobenzaldehyde since they form complex, colored oxidationproducts with the base on heating.

These salts of p-di-alkylaminobenzaldehyde with non-oxidizing inorganicacids may effectively be substituted for the sulphuric acid of Wernerssolution, or the hydrochloric acid of Hartmans reagent, when used inconjunctionwith citric or tartaric acids. These mixtures form stable,solid crystalline compositions and are well adapted to be dispensed inthe form 01 tablets or capsules. The solid organic acids serve not onlyto yield the acid pH on solution in water that is required for thistest, but also stabiland the alkali metals, and the sulphate, nitrateand hydrochloride of urea may also be used as solvents forp-di-alkylaminobenzaldehydes since they will react with the lattergroups of compounds to yield the corresponding water soluble aromaticsalts. These derivatives of urea, however, are too hygroscopic to besatisfactorily included into a solid composition of this nature.

The same is true of bisulphates.

Not only p-dimethylaminobenzaldehyde, but the homologousp-di-alkylaniinobenzaldehydes are equally suitable for the purposes ofthe present invention. Thus, compositions of high sensitivity and goodspecificity may be obtained using p-diethylaminobenzaldehyde (Boessneck,Berichte, 19, 369, 1886). alkylaminobenzaldehydes may be prepared byreacting a di-alkylaniline with formaldehyde andp-nitrosodimethylaniline in strong hydrochloric acid solution to formthe corresponding N, N-dimethyl-N (4-di-alkylaminobenz'al) p-phenylenediamine, and hydrolyzing the latter benzylidene compound (Ullman andFrey, Berichte, 3'7, 858; Adams and Coleman, Organic Syntheses, 2, 17).They form solid, stable bases which are readily suitable forincorporation into the solid compositions of the present invention. Whenadded to an acidified aqueous solution (e. g., diluted urine,deproteinized blood filtrate, cerebrospinal fiuid) containing anaminoarylsulphonamide, they will interact with the latter to form thedeeply yellow colored Schifi bases, of the general formula:

(AlkyllzN- O CII2N- O- somna Thus, for greatest accuracy with thismethod, the

concentration of aminoarylsulphonamide (calculatd as sulphanilamide) inthe deproteinized blood filtrate, cerebrospinal fluid or diluted urineto be tested should lie within the range of 0.1 to 5.0 mm. per cent.

1 find further that the difficulty involved in making a satisfactorycolor comparison of the yellow solution may be obviated by incorporatinga small amount of a water-soluble blue dyestuff into the composition.Inthe absence of aminoarylsulphonamide, these diagnostic compositionsdissolve in the aqueous solution being tested to give a clear bluesolution. In the presence of aminoarylsulphonamide, a green color, isobtained by the combination of the bluev color of the added dyestuff onthe yellow color oi! the Schifl base formed by the interaction of thedialkylaminobenzaldehyde with the aminoarylsulphonamide. The green colorso formed is strictly proportional in intensity to the concentration ofaminoarylsulphonamide in solution In general, these diwithin the abovementioned range of 0.1 to 5.0 mgm. per cent. As is well known to personsskilled in the art, green is a very satisfactory color for colorimetriccomparisons. A color chart comprising a graduated series of color panelscorresponding to increasing concentrations-oi. aminoarylsulphonamide canreadily be made which will enable the physician or technician to make arapid, visual colorimetric comparison with a good degree or accuracy.Examples oi typical water-soluble blue dyestufl's that may be used withgood results in the diagnostic compositions of the present inventionare: Methylene blue, Wright's stain, Patent blue, Victoria blue,indulin, Mel'dolas blue, New blue, alizarin saphirole, indigo carmine,etc. These dyestuiis may be added to the solid diagnostic compositionsin amounts varying from 0.004% to 0.040%, although this range is by nomeans critical and may'vary widely with different dyes. Good results areobtained with Methylene blue in amounts from 0.005% to 0.015% of thetotal weight oi the diagnostic composition.

The factor that contributes most importantly to the sensitivity of thediagnostic compositions of this invention is the purity of thedi-alkylaminobenzaldehyde. All "technical and most so-called "puriiiedgrades of p-d methylaminobenzaldehyde made by the method of Ullman andFrey vary in color from a light to a deep lemon yellow. It is obvious,of course, that a yellow colored reagent will interfere with thedetection of small amounts of aminoarylsulphonamides. Diagnosticcompositions containing impure, slightly coloredp-dimeth'ylaminobenzaldehyde will yield light green blanks" withdistilled water, rather than the true blue of the added'dye. It isimportant, therefore, that an absolutely colorless grade ofp-dimethylaminobenzaldehyde be used in these diagnostic compositions. Itis possible, of course, to use slightly colored grades ofp-dimethylaminobenzaldehyde in the compositions of the presentinvention; but when this is done his necessary to run a blank" withdistilled water through which the color panels are observed when thetest specimen is compared. This procedure, however, is not recommendedwhen the pure p-dimethylaminobenzaldehyde is available.

Technical p-dimethylaminobenzaldehyde may be freed or its undesirablecolor by the method described by Adams and Coleman (Organic Synthesis,II, 19). 75.0 grams (0.5 mole) of p-dimethylaminobenzaldehyde isdissolved with stirring in a solution of 60 cc. (0.725 mole) of 23 36.hydrochloric acid and 360 cc. of water. The

solution is diluted with 200 cc. of water and filtered i'rominsolublematter. To the vigorously agitated filtrate is then added slowly 65 cc.of a 20% sodium hydroxide solution. The precipitate ofp-dimethylaminobenzaldehyde that forms carries down with it the majorportion of the undesired coloration. This is filtered off and to thefiltrate is then added slowly with vigorous mixing. 76 cc. of 20% sodiumhydroxide solution. The major portion of the aldehyde is thusprecipitated as a mass of colorless crystals which are filtered off,washed with water and dried at 30- 40 C. in an incubator. To thefiltrate from this precipitation there is then added 3 cc. of 20% sodiumhydroxide solution, The slightly colored precipitate that forms isfiltered of! and added to the first precipitate of impure material. Thecombined precipitates are then worked up 4 g with the next batch ofcrude material. About 57.0 to 58.0 grams of colorlessp-dimethylaminobenzaldehyde is thus obtained per batch. The over-allconversion of the colored, technical grade of aldehyde to the purified,colorless compound is 90 to 95 per cent.

The diagnostic compositions of this invention may be used to detect andestimate all aminoarylsulphonamides of the general formula:

The color produced by any given amount of, say, sulfanilamide, isequivalent in intensity to that produced by an equivalent molality ofsulphapyridine, sulphathiazole, sulphadiazine, etc. Thus, a color panelrepresenting a sulphanilamide cncentration of 10.0 mgm. per cent willlikewise represent 14.'7 mgm. per cent of sulphapyridine, 14.8 mgm. percent of sulphathiazole, 20.7 mgm. per cent of sulphanilylsulphanilamide,etc. (Lankford, American Journal of Clinical Pathology TechnicalSupplement, 4, 155, 1940). Similarly, a color panel representing aconcentration of 10.0 mgm. per cent of sulphanilamide in blood filtratethat represents a 1:10 dilution of the original whole blood will alsorepresent a sulphanilamide concentration of 200.0 mgm. per cent in urinediluted 1:200. Each color panel in the color chart may conveniently beara legend interpreting the intensity of that color in terms of blooddiluted 1:10 and urine diluted 1:200, and in terms of each of theaminoarylsulphonamides that may be tested by this method, thus:

Blood l:l0 Urine 1:200

lllgm. per cefll ler cm! Sulphanilamlde 10. u o. 200 Sulphapyridlne l4.7 n. 294 Sulphathiazolc 14. 8 0. 25m

Etc. etc. etc.

The following examples of typical compositions covered by this inventionare given to define and illustrate the present invention, but in no wayto limit it to the reagents, proportions, or conditions describedtherein. Obvious modifications will occur to any person skilled in theart.

Example 1 Eight grams of pure, colorless p'-dimethylaminobenzaldehyde isdissolved in 10 cc. of concentrated (36%) hydrochloric acid and istriturated with 450.0 grams of anhydrous granular citric acid and 25.0cc. of a 0.2% alcoholic solu- Seventeen and one-half grams ofp-diethylaminobenzaldehyde are dissolved in the smallest quantity of hot90%"-ethanol, and 5.5 grams of concentrated sulphuric acid diluted withan equal weight of ethanol is added slowly thereto. The

alcohol is distilled oil and the solid residue ofp-diethylaminobenzaldehyde sulphate is dried and powdered. Ten grams ofthedried p-diethylaminobenzaldehyde sulphate, 460.0 grams of tartaricacid, milligrams of medicinal methylene blue, and 30 grams of sodiumbicarbonate are triturated together until a completely homogeneousmixture is obtained. This mixture is then compressed into five-graintablets or' dispensed in five-grain capsules.

In the above examples the function of the carbonate is to cause rapiddisintegration of the tablet by efiervescence when placed in thesolution to be tested.

Procedure for urine A five grain tablet or capsule of the abovecomposition is dissolved in 2.0 cc. of tap water in a test tubegraduated at 10 cc. After solution is complete, 0.05 cc. of the urine tobe tested is added, the mixture is diluted to the 10.0 cc. mark withwater, corked and mixed for a few seconds. The color that forms almostinstantly is then compared with a color chart.

Procedure for blood An excellent composition for effecting thedeproteinization of whole blood may be obtained by mixing:

- Grams Sodium tungstate (Na2WO42H2O) 357.5 Citric acid monohydrate 94.0Sodium bicarbonate 111.0 Sodium bisulphate 437.5

until homogeneous. The mixture is then compressed into 2.5 grain tabletsor dispensed in 2.5 grain capsules. lactose or talc may be added tofacilitate the formation of the tablets provided the relativeproportions and absolute amounts of the above compounds remain the samein each tablet.

The test may conveniently be carried on in a test-tube graduated at 3.5cc. and 5.0 cc. A 2.5 grain tablet or capsule of the protein precipitantcomposition is placed in the tube and 2 to 3 cc. of water added. Whenthe tablet has dissolved, the solution is diluted to the 3.5 cc. mark.If talc has been used as an excip'ient or binder in the tablet, thesolution will be turbid; otherwise a clear solution is obtained.

Exactly 0.5 cc. of the blood to be tested (oxalated or citrated, ifpreserved) is now added dropwise to the solution while gently shakingthe tube back and forth. After the blood has been added, the tube isallowed to stand for five 'paper. The filtrate is collected in a clean,dry,

test tube and compared with a color chart for intensity.

At this point it should be stressed that the blue dyestufi employed inmy diagnostic compositions must be the same as that used in thepreparation of the color chart with which the final colored solution isto be compared. I prefer to use medicinal methylene blue since it is awell standardized product, and samples from difi'erent batches from themanufacturer can be relied upon to give the same coloration at a givenconcentration. The color panels should be made to Diluents or excipientssuch as v 10. A process for testing for aminoarylsulphoncorrespond tosolutions of my diagnostic tablets with certain concentrations oi theaminoarylsulphonamides, the blue dyestufl of the tablets beingcontrolled so as to give an isochromatic appearance at any givenconcentration of aminoarylsulphonamide.

Having described my invention, what I claim and desire to protect byLetters Patent is:

1. Diagnostic compositions comprising in dry solid form a member of thegroup consisting of the salts of the para-dialkylaminobenzaidehydes withnon-oxidizing inorganic acids, a member of the group consisting ofcitric and tartaric acids. and a water-soluble blue dyestuif.

2. Diagnostic compositions comprising in dry solid forma member of thegroup consisting of the salts of para-dimethylaminobenzaldehyde withnon-oxidizing inorganic acids, a member of the group consisting ofcitric and tartaric acids, and a water-soluble blue dyestuif.

3. Diagnostic compositions comprising in dry solid formpara-dimethylaminobenzaldehyde hydrochloride, a member of the groupconsisting of citric and tartaric acids, and methylene blue.

4. Diagnostic compositions comprising in dry solid formpara-dimethylaminobenzaldehyde hy- 8. Diagnostic compositions comprisingin dry solid form a substantially colorless grade ofparadimethylaminobenzaldehyde hydrochloride, citric acid, methylene blueand sodium bicarbonate.

9. A process for testing for aminoarylsulphonamides in fluids whichconsists in adding to a measured amount of said fluid a measured amountofa composition as described in claim 1, to produce a color which bycomparison with a color standard indicates the quantity ofaminoarylsulphonamid in the fluid. y

amides in body fluids which consists in adding to a predetermined amountof the body fluid, a predetermined amount of a fixed proportion mixturecontaining in dry solid form a member of the group consisting of thesalts of the para-dialkylaminobenzaldehydes with non-oxidizing inorganicacids, a member of the group consisting of citric and tartaric acids anda water-soluble blue dyestuif, to produce a color and comparing saidcolor with predetermined standard colors to indicate the percentage ofaminoarylsulphonamide in the body fluid.

11. A process for testing a liquid containing an aminoarylsulphonamideto determine the quantity thereof, which comprises dissolving in water adry solid composition containing a predetermined quantity of astandardized blue color soluble in the resulting solution; containing asalt of p-dialkylaminobenzaldehyde with one of the non-oxidizinginorganic acids, which salt is in excess of that reactive with themaximum amount of aminoarylsulphonamide compound to be determined; andcontaining a member of the group consisting of citric and tartaric acidsin quantity to effect solution of the salt ofp-dialkylaminobenzaldehyde; adding a predetermined quantity of theliquid to be tested and efiecting a volume for the solutionpredetermined by a prescribed control of the test, whereby a yellowcolor is formed in proportion to the amount of aminoarylsulphonamidepresent, and whereby said yellow color with the said blue color gives agreen color suitable for comparison with a green color chart calibratedfor the test in terms of variable quantities of aminoarylsulphonamidedetermined by the test.

12. Diagnostic compositions comprising in dry solid form apara-dialkylmino-benzaldehyd hydrochloride, a member oi the groupconsisting of citric and tartaric acids, and a water-soluble bluedyestuil.

13. Diagnostic compositions comprising in dry solid formpara-dimethylamino-benzaldehyde hydrochloride, a member of the groupconsisting of citric and tartaric acids, and a water-soluble bludyestufi.

14. Diagnostic compositions comprising in dry solid form apara-dimethylamino-benzaldehyde salt with one of the non-oxidizinginorganic acids, a member of the group consisting of citric and tartaricacids, and methylene blue.

JONAS KAMLET.

